70–120 mg tissues were lysed in 5 μL/mg tissue RIPA buffer (Sigma) with 1× Complete protease inhibitors and 1 U/μL Superase In RNase inhibitor (Thermo Scientific) using Fisherbrand Pellet Pestle Cordless Motor. Afterwards, homogenization debris was removed by centrifugation at 20.800 × g for 10 min at 4°C. Protein concentration was determined using APA assay (Cytoskeleton Inc) and 50 μg total protein were separated on SDS-PAGE followed by western blot. Membranes were stained with anti-Naa10, anti-Naa15, and anti-GAPDH antibodies (all Abcam).
For RNA purification, 30 μL clarified lysates were mixed with 70 μL RNase free water and RNA isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers recommendations, including on-column Dnase digest. 1 μg RNA was reverse transcribed using the TaqMan Reverse transcription kit and gene level detection performed using SYBR Green Master Mix (all Thermo Scientific). Relative expression was normalized to GAPDH and ACTB.
For the characterization of the mNaa12 antibody, tissue was lysed in 2 µL per mg tissue PBS-X (PBS + 0.2% [v/v] Triton X-100 + 1× Complete protease inhibitor cocktail). 10–200 µg lysate were subjected to SDS-PAGE and western blot.
For RNA purification, 30 μL clarified lysates were mixed with 70 μL RNase free water and RNA isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers recommendations, including on-column Dnase digest. 1 μg RNA was reverse transcribed using the TaqMan Reverse transcription kit and gene level detection performed using SYBR Green Master Mix (all Thermo Scientific). Relative expression was normalized to GAPDH and ACTB.
For the characterization of the mNaa12 antibody, tissue was lysed in 2 µL per mg tissue PBS-X (PBS + 0.2% [v/v] Triton X-100 + 1× Complete protease inhibitor cocktail). 10–200 µg lysate were subjected to SDS-PAGE and western blot.